The invention relates to identification and isolation of the simple sequence repeat (SSR) loci in the higher eukaryotes, such as the plants, and particularly the pines. The SSR loci of the invention are particularly useful as genetic markers for genetic mapping, population genetics studies and inheritance studies in various plant breeding programs.
Loblolly pine (Pinus taeda L.) is an important, experimental and commercial forest tree species native to the southeastern United States. Loblolly pine is planted extensively in the southeastern United States and to lesser degrees in other warm temperate regions of the world. In the United States, plantations are managed and utilized for a variety of products including raw materials (wood, fiber, and chemicals), ecosystem components (wildlife habitat and water and soil conservation), and recreational activities. Most of planting stock originates from production seed orchards established by various loblolly pine improvement programs. To date, such programs have completed one to three cycles of selection using progeny testing for parental selection and seed orchard development, and family and within-family testing and selection for population improvement. Loblolly pine breeding has various limitations, such as, long generation times to flower ( greater than 5 years) and harvest ( greater than 15 years), low tolerance to inbreeding, large size of individual trees, variable sites for testing and replanting, difficulty of vegetative propagation, low heritability of important traits, and uncertainty of trait values.
Marker-assisted selection (MAS) using DNA-based markers has much potential for improving the efficiency and effectivenes of tree breeding programs (O""Malley and McKeand 1994 For. Genet. 1:207-218.). Important improvements afforded by MAS include reducing the time-to-selection and improving the accuracy of selection. An important goal of such research is to identify DNA markers or other measures that predict performance of mature trees. With this information, tree breeders could more confidently select trees at an early age, induce them to flower, and breed them to produce the next generation. In addition, selections made at an early age could be vegetatively propagated in mass using rooted cutting or tissue culture based technologies (Bradshaw and Foster 1992 Can. J. For. Res., 22:1044-1049.). Vegetative propagation and deployment has the potential to greatly improve wood and fiber yield and quality by capturing within-family genetic variation and providing better performing varieites for plantation establishment.
Several of the fundamental limitations to MAS applications in loblolly pine (Strauss et al. 1992 Can. J. For. Res., 22:1050-1061.) have been overcome in recent years. Most notably is the application of randomly-primed, PCR-based genetic markers (e.g., RAPD) to parent- or family-specific genome mapping (Tulsieram et al. 1992, Biotechnology, 10:686-690; Nelson et al 1994 Journal of Heredity, 85:433-439; Plomion et al. 1996 Theor. Appl. Genet., 93:1083-1089., Wilcox et al. 1996 Proc. Natl. Acad. Sci. USA, 93:3859-3864.). Although family-specific mapping and MAS approaches have potential, these methods are limited to situations where small breeding ( less than 10 parents) populations are maintained with progeny established in large-family (n greater than 500) tests. In practice, however, most loblolly pine breeding programs do not fit this situation. More typical is large breeding populations, sometimes several populations per program, and always relatively small-family (n less than 150) progeny tests. In addition most programs now include many pedigrees of at least three-generations, with nearly mature third-generation trees in the field. Utilizing existing extensive pedigree and progeny test information is essential for developing better MAS technology and improving breeding programs.
Currently available marker systems are not optimal for detecting QTL variation across families and across multi-generation pedigrees. Reviews of current marker technologies and their limitations to use in QTL mapping and MAS is provided by Neale and Harry (1994 AgBiotech News Info., 6:107N-114N.) and O""Malley and Whetten (1997 Molecular markers and forest trees. DNA Markers: Protocols, Application and Overviews ed. G. Caetano-Anollxc3xa9s and P. M. Gresshoff. John Wiley and Sons, New York., 237-257.). Given a genome size of about 2000 cM(K) for loblolly pine, a large number of highly polymorphic, co-dominant genetic markers will be needed for genome mapping and QTL analyses (Echt and Nelson 1997 Theor. Appl. Genet., 94:1031-1037.).
Accordingly, there is a need in the art for new genetic markers. In an effort to develop such markers for loblolly pine, the pines and the plants in general, the present inventors developed simple sequence repeat (SSR) markers described herein. The markers of the invention are also useful for other eukaryotic organisms.
Simple sequence repeats (SSRs), which are also known as microsatellite DNA repeats, have now been discovered in the pines and have been shown to exhibit length polymorphisms. These repeats represent an abundant pool of potential genetic markers.
Accordingly, in one aspect, the present invention relates to the plant SSR motifs, such as for example, di-, tri- and tetra-nucleotide repeated motifs.
In another aspect, the invention relates to the polynucleotides containing one or more such SSR motifs and the primers for the amplification of the fragments containing SSRs. The primers may be cloned polynucleotide fragments or chemically synthesized oligonucleotides, and contain at least a portion of the non-repeated, non-polymorphic sequence fang SSRs on either 5xe2x80x2 or 3xe2x80x2 end.
The present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymorphisms of the type described in this application The kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.
In a further aspect, the invention provides for a method of analyzing one or more specific SSR polymorphisms in an individual or a population, which involves amplification of small segment(s) of DNA containing the SSR and non-repeated flanking DNA by using the polymerase chain reaction, and sizing the resulting amplified DNA, preferably by electrophoresis on polyacrylamide gels.
In yet another aspect, the invention provides for a method of determining the sequence information necessary for primer production by isolation and sequencing of DNA fragments containing the SSRS, using hybridization of a synthetic, cloned, amplified or genomic probe, containing sequences substantially homologous to the SSR, to the DNA.
In a further aspect, the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant. The method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymorphic amplified DNA marker containing one or more SSR sequences.
In yet another aspect, the SSR markers of the invention are used in commercial plant breeding. Traits of economic importance in plant crops can be identified through linkage analysis using polymorphic DNA markers.
All patents, patent applications and references cited in this specification are hereby incorporated herein by reference in their entirety. In case of any inconsistency, the present disclosure governs.
Definitions
The following terms and phrases are used throughout the specification with the following intended meanings.
The abbreviation xe2x80x9cSSRxe2x80x9d stands for a xe2x80x9csimple sequence repeatxe2x80x9d and refers to any short sequence, for example, a mono-, di-, tri-, or tetra-nucleotide that is repeated at least once in a particular nucleotide sequence. These sequences are also known in the art as xe2x80x9cmicrosatellites.xe2x80x9d A SSR can be represented by the general formula (N1N2 . . . Ni)n, wherein N represents nucleotides A, T, C or G, i represents the number of the nucleotides in the base repeat, and n represents the number of times the base is repeated in a particular DNA sequence. The base repeat, i.e., N1N2 . . . Ni, is also referred to herein as an xe2x80x9cSSR motif.xe2x80x9d For example, (ATC)4, refers to a tri-nucleotide ATC motif that is repeated four times in a particular sequence. In other words, (ATC)4 is a shorthand version of xe2x80x9cATCATCATCATC.xe2x80x9d
The term xe2x80x9ccomplement of a SSR motifxe2x80x9d refers to a complementary strand of the represented motif. For example, the complement of (ATG) motif is (TAC).
The term xe2x80x9cpermutations of a SSR motifxe2x80x9d refers to all possible combinations of a motif found within the repeated sequence of that motif. For example, permutations of the (ATG)5 motif (i.e., ATGATGATGATGATG) are TGA and GAT as both can be found in this repeat.
The term xe2x80x9cperfect repeatxe2x80x9d refers to a repeated SSR motif without interruption and without adjacent repeat(s) of a different motif. However, the repeats may be xe2x80x9cimperfectxe2x80x9d when a repeated SSR motif is interrupted by a number of non-repeated nucleotides, such as for example in (AC)5GCTAGT(AC)7. Other possible variations of SSRs would be known to those of skill in the art. These repeats, including compound repeats, are defined by Weber, J. L., 1990, Genomics, 7:524-530.
The term xe2x80x9ccompound repeatxe2x80x9d refers to a SSR that contains at least two different repeated motifs that may be separated by a stretch of non-repeated nucleotides. An example of a compound repeat is (ATC)5(AT)6.
The term xe2x80x9cSSR locusxe2x80x9d refers to a location on a chromosome of a SSR motif; locus may be occupied by any one of the alleles of the repeated motif. xe2x80x9cAllelexe2x80x9d is one of several alternative forms of the SSR motif occupying a given locus on the chromosome. For example, the (ATC)8 locus refers to the fragment of the chromosome containing this repeat, while (ATC)4 and (ATC)7 repeats represent two different alleles of the (ATC)8 locus. As used herein, the term locus refers to the repeated SSR motif and the flanking 5xe2x80x2 and 3xe2x80x2 non-repeated sequences. SSR loci of the invention are useful as genetic markers, such as for determination of polymorphysm.
xe2x80x9cPolymorphismxe2x80x9d is a condition in DNA in which the most frequent variant (or allele) has a population frequency which does not exceed 99%.
The term xe2x80x9cheterozygosityxe2x80x9d (H) is used when a fraction of individuals in a population have different alleles at a particular locus (as opposed to two copies of the same allele). Heterozygosity is the probability that an individual in the population is heterozygous at the locus. Heterozygosity is usually expressed as a percentage (%), ranging from 0 to 100%, or on a scale from 0 to 1.
The term xe2x80x9cinformativenessxe2x80x9d is a measure of the utility of the polymorphism. In general, higher informativeness means greater utility. Informativeness is usually defined in terms of either heterozygosity or xe2x80x9cPolymorphism Information Contentxe2x80x9d (PIC) (for PIC see Botstein, D., et al., 1980, Am. J. Hum. Genet., 32, 314-331). The PIC represents the probability that the parental origin of an allele can be determined from the marker genotype of the locus in any given offspring. The PIC values range from 0 to 1.0, and are smaller in value than heterozygosities. The formulas for calculating H and PIC are disclosed in the examples. For markers that are highly informative (heterozygosities exceeding about 70%), the difference between heterozygosity and PIC is slight.
xe2x80x9cPrimersxe2x80x9d are short polynucleotides or oligonucleotides required for a polymerase chain reaction that are complementary to a portion of the polynucleotide to be amplified. The phrase xe2x80x9cprimer adapted for detection of a SSR markerxe2x80x9d means that the primer is capable of amplyfying a particular SSR locus to be used as a marker, wherein the primer is complementary to either the 5xe2x80x2 or the 3xe2x80x2 non-repeated region of the SSR locus and is of a length suitable for use as a primer. For example, the primer is no more than 50 nucleotides long, preferably less than about 30 nucleotides long, and most preferably less than about 24 nucleotides long.
The term xe2x80x9cpolynucleotidexe2x80x9d is intended to include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense strands together or individually (although only sense or anti-sense stand may be represented herein). This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as xe2x80x9cprotein nucleic acidsxe2x80x9d (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
An xe2x80x9cisolatedxe2x80x9d nucleic acid or polynucleotide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide may contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. A polynucleotide amplified using PCR so that it is sufficiently and easily distinguishable (on a gel from example) from the rest of the cellular components is considered xe2x80x9cisolatedxe2x80x9d. The polynucleotides of the invention may be xe2x80x9csubstantially pure,xe2x80x9d i.e., having the highest degree of purity that can be achieved using purification techniques known in the art.
The term xe2x80x9chybridizationxe2x80x9d refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing.
Polynucleotides are xe2x80x9chybridizablexe2x80x9d to each other when at least one strand of one polynucleotide can anneal to a strand of another polynucleotide under defined stringency conditions. Hybridization requires that the two polynucleotides contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5xc3x97SSC at 65xc2x0 C.) requires that the sequences exhibit some high degree of complementarily over their entire sequence. Conditions of intermediate stringency (such as, for example, an aqueous solution of 2xc3x97SSC at 65xc2x0 C.) and low stringency (such as, for example, an aqueous solution of 2xc3x97SSC at 55xc2x0 C.), require correspondingly less overall complementarily between the hybridizing sequences. (1xc3x97SSC is 0.15 M NaCl, 0.015 M Na citrate.) As used herein, the above solutions and temperatures refer to the probe-washing stage of the hybridization procedure. The term xe2x80x9ca polynucleotide that hybridizes under stringent (low, intermediate) conditionsxe2x80x9d is intended to encompass both single and double-stranded polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide.
The term xe2x80x9c% identityxe2x80x9d refers to the percentage of the nucleotides of one polynucleotide that are identical to the nucleotides of another sequence of identical length (excluding the length of the SSR) as implemented by the National Center for Biotechnology Information. The % identity value may be determined using a PowerBlast program (Zhang and Madden 1977 Genome Res. 7:649-56.).
The term xe2x80x9c% homologyxe2x80x9d between the sequences is a function of the number of matching positions shared by two sequences divided by the number of positions compared and then multiplied by 100. This comparison is made when two sequences are aligned (by introducing gaps if needed) to give maximum homology. PowerBlast program, implemented by the National Center for Biotechnology Information, is used to compute optimal, gapped alignments. Alternatively, the % homology comparison may be determined using a Blast 2.0 program implemented by the National Center for Biotechnology Information.
SSR Motifs and SSR Loci of the Invention
The present invention relates to SSR motifs and SSR loci useful as genetic markers in various organisms, particularly plants. In a preferred embodiment of the invention, the SSR motifs and loci originate from the pines, such as the pines of the Pinus genus, for example P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, and P. sylvestris. As seen from the list of exemplary species, the pines and SSRs thereof of the present invention can belong to either of the two subgenera of the Pinus genus. P. strobus (white pine) is a species of the Strobus subgenus, and P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, and P. sylvestris are exemplary species of the Pinus subgenus.
The SSR motifs of the invention have the general formula (N1, N2 . . . Ni)n, wherein: N represents nucleotides A, T, C or G; i represents the number of the last nucleotide in the SSR motif; and n represents the number of times the SSR motif is repeated in the SSR locus. In one embodiment of the invention, the total number of nucleotides in a motif (i) is about six, preferably four, three or two. The total number of repeats (n) may be from 1 to about 60, preferably from 4 to 40, and most preferably from 10 to 30 when i=2; preferably 4-25, and most preferably 6-22 when i=3; and preferably 4-15, and most preferably 5-10 when i=4. Any SSR motif of the above formula is within the scope of the invention, however, the following SSR motif are preferred: AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC, AAAC, AAAT, AGAT and all complements and permutation of said motifs, such as for example ATG, CAT, TTG, TTA, TTC, ATT, and TAT. Compound repeats are also within the scope of the invention. Examples of such repeats are: (A)n . . . (ATG)n; (ATG)n . . . (C)n; (CAT)n . . . (A)n; (ACC)n . . . (ATC)n; (TTG)n; . . . (TTA)n; (C)n . . . (ATT)n; (TAT)n . . . (A)n; (ATT)n . . . (AAT)n; (TTC)n . . . (T)n; and (A)n(AAAC)n(A)n.
The SSR loci of the invention are preferably a maximum about 500 nucleotides long. In another preferred embodiment, the SSR locus of the invention is a minimum of 50 nucleotides long.
The invention further provides for isolated polynucleotides comprising at least one SSR motif and having the nucleotide sequences as shown in Table 3 (SEQ ID NOS: 237 to 354). These polynucleotides may be of the same length as the sequences shown in Table 3 or alternatively comprise additional sequences on their 5xe2x80x2, 3xe2x80x2 or both ends. The latter polynucleotides may be less than about 500 bp, less than about 1 kb, less than about 2 kb or less than about 3 kb long. In an embodiment of the invention, the polynucleotides comprising the sequences of SEQ ID NOS: 237-354 do not containing any functional gene or coding sequences.
Further within the scope of the invention are polynucleotides that (i) hybridize under the conditions of low, medium or high stringency to the polynucleotides comprising the sequences of SEQ ID NOS: 237-354 and (ii) contain SSR motifs. In certain embodiment of the invention, these hybridizable polynucleotides are less than about 1000 bp long, more preferably less than about 500 bp long and most preferably less than about 200 bp long. In one embodiment of the invention, the hybridizable polynucleotide is about the same length as the polynucleotide to which it hybridizes.
Also within the scope of the invention are polynucleotides that contain SSR motifs and have at least about 75%, preferably at least about 85%, and most preferably at least about 95% identity to the polynucleotides having the sequence of SEQ ID NOS:237 to 354.
Polynucleotides that contain SSR motifs and have at least about 75%, preferably at least about 85%, and most preferably at least about 95% homology to the polynucleotides having the sequence of SEQ ID NOS:237 to 354 are also within the scope of the invention.
In one preferred embodiment of the invention, polynucleotides that align to polynucleotides of SEQ ID NO:237-354 under the following conditions are also within the scope of the invention: alignment done using PowerBlast network client on PowerMacG3, when the search is set to high stringency (M=1, N=xe2x88x925, S=80, S2=80) for blastn, without gap alignment. Most preferably, these polynucleotides are not of human origin.
In another preferred embodiment of the invention, polynucleotides that align to polynucleotides of SEQ ID NO:237-354 under the following conditions are also within the scope of the invention: alignment done using either PowerBlast or Blast 2.0 program using the following parameters: match=1, mismatch=xe2x88x922, gap open=5, gap extension=2, x_dropoff=50, expect=10, and wordsize=9. Most preferably, these polynucleotides are not of human origin.
Isolated polynucleotides comprising at least one SSR motif and having the property of being amplifiable from a genomic DNA using PCR and any of the primer pairs disclosed in Tables 2 and 7 are also within the scope of the invention. These polynucleotides may be identified and isolated by amplification of any genomic DNA. Prefereably, genomic DNA used is a plant DNA, more preferably the pine DNA and most preferably the DNA from the Pinus genus. For example, genomic DNA may be from P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, or P. sylvestris. In one embodyment of the invention, these polynucleotides are less than about 500 bp long. However, the length of the amplified DNA fragment is generally limited only by the resolving power of the particular separation system used. The thin denaturing gels, for example, are capable of resolving fragments differing by as little as 1 base up to a total fragment length of about 300 bp. Use of longer gels and longer electrophoresis times can extend the resolving power up to about 600 bp or more. However, the longer the fragment, the lower the proportion of its length is occupied by the SSR sequences, and hence the resolution is more difficult.
Oligonucleotide primer adapted for detection of SSR marker are also within the scope of the invention. A suitable primer comprises at least the sequence of SEQ ID NOS:1-236 and 367-390.
The present invention also provides probes specific to at least part of the aforesaid SSRs for detecting SSR markers using methods other than polymerase chain reaction, such as for example hybridization with labeled probes. The probes useful in the invention may be any sequence comprising at least the sequence of SEQ ID NOS: 1-236, as well as any other probe that a person of skill in the art can construct based on the information of SEQ ID NOS: 237-354.
The SSR loci of the invention may be polymorphic. They may have a PIC of at least 30% (0.3); preferably of at least 70% (0.7); and most preferably of at least 90% (0.9).
The polynucleotides and primers of the invention may be subcloned and introduced into various host cells according to methods well known in the art. The resulting clones and host cell are also within the scope of the invention. A person of skill in the art can make all such constructs and host cells using methods known in the art. However, the following non-limiting examples are provided below.
A large number of vectors, including bacterial, fungal and plant vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech, Palo Alto, Calif.), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), or pRSET or pREP (Invitrogen, San Diego, Calif.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.
Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl2 mediated DNA uptake, fungal infection, microinjection, microprojectile transformation, or other established methods. Appropriate host cells include bacteria, archaebacteria, fungi, especially yeast, and plant and animal cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, and the like.
The present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymorphisms of the type described in this application. The kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.
Development and Use of Polymorphic DNA Markers
The present invention provides for the methods of identifying and isolating SSR loci and their use as genetic markers.
In one embodyment, a method for the identification from genomic DNA of a fragment comprising a SSR locus comprising the steps of: (i) contacting a DNA library with at least one hybridisation probe so as to identify a population of DNA fragments enriched for simple tandem repeats; (ii) isolating and cloning said population; and (iii) screening of the resulting DNA library so as to identify an individual fragment comprising a simple tandem repeat locus.
The DNA library may be a genomic DNA library; the genomic DNA library may be any convenient population of DNA fragments such as pine DNA, or subgenomic DNA libraries such as those generated by PCR from flow sorted chromosomes (see Telenius, H., et al., 1992, Genomics 13: 718-725). The genomic DNA library may be obtained by restriction digestion of genomic DNA. The average fragment size within the DNA library may be less than 1.5 kilobases and may be less than about one kilobase. The fragment size may be from about 400 bp to about 1000 bp.
The hybridisation probe or set of probes may be immobilised on a solid phase such as a nylon membrane and may be used to identify a particular class of SSRS. Such classes may include dimeric, trimeric, tetrameric, pentameric and hexameric repeats. Particular oligonucleotide probes for use in the present invention may include oligonucleotide probes comprising a repeated region of greater than 200 bp. The probe may comprise repeats having at least 70%, such as 85% or 100%, identity to a given repeat sequence. The hybridisation probe may be a set of probes comprising mixed trimeric or tetrameric repeat DNA or any other combination of various SSR motifs.
The population of DNA fragments enriched for SSR may be amplified prior to cloning and this may be effected by PCR amplification. Universal linker sequences may be ligated to the ends of individual fragments, possibly prior to the enrichment procedure, and linker sequence specific primers may then be used to amplify the enriched population. Linker sequences may then be removed, for example by restriction digestion, prior to cloning.
In another embodiment, a method for the identification from genomic DNA of a fragment comprising a SSR locus comprises the steps of: (i) ligating universal linker sequences to the ends of fragments comprised in a genomic DNA library so as to form a library for PCR amplification; (ii) contacting said PCR library with at least one hybridisation probe so as to identify a population of library fragments enriched for simple tandem repeats; (iii) separating and amplifying said population by PCR; and (iv) cloning and screening the resulting amplification products so as to isolate an individual fragment comprising a simple tandem repeat locus.
Cloning may be effected using any convenient cloning procedure and vector (for example pBluescriptII (Stratagene, Lajolla, Calif.)) such as those described by Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press.
Screening may be effected using any convenient hybridisation probe or set of probes comprising SSR sequences. These may be the same as those disclosed above in respect of the enrichment procedure.
A more detailed description of possible ways of detecting SSR loci is provided in the Examples.
Individual clones comprising SSR loci may be analyzed using conventional techniques to determine, for example, specific sequence information. Such techniques may allow the generation of individual xe2x80x9cidentitiesxe2x80x9d specific for one or more polymorphic loci. The generation of such individuals xe2x80x9cidentitiesxe2x80x9d may be used to identify and characterise family relationships and may be used for e.g. trait tracing in a breeding program and in any other technique which uses SSRs and their polymorphisms.
According to a further aspect of the present invention there are also provided methods of genetic characterisation wherein sample DNA is characterised by reference to at least one of the SSR loci, primer sequences and probes of the invention. The method of genetic characterisation may comprise either the use of at least one hybridisation probe or it may comprise the use of polymerase chain reaction (PCR) primers specific to at least one of the SSR loci in order to amplify selectively the SSR locus. The PCR primers may comprise at least one of the primers and probes of the present invention. The method of genetic characterisation may be used in genetic mapping studies such as linkage studies, and may be used in the genetic analysis of inherited traits.
In one embodyment, the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant. The method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymorphic amplified DNA marker containing one or more SSR sequences. The analysis comprises amplification using the polymerase chain reaction of one or more short DNA fragments containing the SSR followed by measurement of the sizes of the amplified fragments using gel electrophoresis.
Examples of using SSR markers of the invention for detection of polymorphism in various pines are provided in the Examples. Any other known uses of such markers will be apparent to persons of skill in the art.
Throughout the present application, the standard IUPAC nucleotide representation was used. It should be noted that in these, K=G or T (keto); Y=C or T (pYrimidine); R=A or G (puRine); M=A or C (aMino); S=G or C (strong 3H bond); B=C, G or T; D=A, G or T; H=A, C or T; and V=A, C or G.
The present invention is further described in the following non-limiting examples.